The present invention relates to a novel advantageous process for amplifying nucleic acids using DNA/PNA primers and a temperature-stable polymerase enzyme.
Recently, polyamide-nucleic acid derivatives (PNAs) have been described (Michael Egholm, Peter E. Nielsen, Rolf H. Berg and Ole Buchardt, Science (1991) 254, 1497-1500; WO 92/20702; M. Egholm et al. Nature (1993) 365, 566-568; P. Nielsen, Bioconjugate Chem. (1994) 5, 3-7) which bind to complementary target sequences (DNA or RNA) with a higher affinity than natural oligonucleotides. In these so-called PNAs, the deoxyribose phosphate structure is replaced by a polyamide oligomer.
It has been found that PNAs of DNA polymerases are not accepted as primers (H. .O slashed.rum, et al., Nucl. Acids Res. (1993) 21, 5332-5336; D. B. Demers, et al., Nucl. Acids Res. (1995) 23, 3050-5) and thus cannot be used as primers for amplifying nucleic acids with the aid of DNA polymerases.
In EP-A 0672 677 and WO 95/08556, PNA/DNA hybrid molecules are described which can also be employed, inter alia, as primers for amplifying nucleic acids using Klenow polymerases.
An advantage of the use of PNA/DNA primers instead of natural oligonucleotide primers for amplifying nucleic acids is that the nucleic acid strand copied with the aid of the PNA primer contains, at the 5' end, a PNA moiety which is stable to 5'-exonucleases. All natural DNA and RNA sequences in the reaction mixture can thus be degraded by 5'-exonucleases without the PNA-containing strand being attacked. However, the polymerase reactions with Klenow polymerase enzymes described in WO 95/08556 only succeed if the PNA/DNA primers described therein have at least four nucleotides at the 3' terminus, while a PNA/DNA primer with only two nucleotides at the 3' terminus fails in the DNA polymerase reaction.
It is known from EP-A 0672 677 that PNA/DNA hybrids with only one 5'-deoxy-5'-aminonucleoside at the carboxyl terminus can be employed as primers if the DNA polymerase of E. coli (Klenow fragment) is employed for the enzymatic polymerization.
These results are completely unexpected, since it is evident from crystal structure studies on DNA polymerase/primer-template complexes that interactions between the phosphodiester functions of the primer and the enzyme exist in several positions (C. M. Joyce, T. A. Steitz, Annu. Rev. Biochem. (1994) 63, 777-822; S. H. Eom, Nature (1996) 382, 278-281).
The fact that the elimination of the negative charges in the primer counteracts the acceptance of the primer by polymerases was additionally confirmed by investigations with primers in which the natural phosphodiester functions were partially replaced by neutral internucleoside bridges, such as, for example, 3'-O-sulfonate or 3'-N-sulfonamide (K. A. Perrin et al., J. Am. Chem. Soc. (1994) 116, 7427-7428).
On the basis of these results, it was to be assumed that with respect to its partial acceptance of uncharged primers, the Klenow enzyme, which is constituted by the PNA/DNA hybrid molecules described in EP-A 0672 677 and with a 5'-deoxy-5'-aminonucleoside at the carboxyl terminus, is an exception among the various DNA polymerases.
A disadvantage of the use of the Klenow polymerase for amplifying nucleic acids using PNA/DNA primers is that this enzyme is not sufficiently temperature-stable that, for example, an amplification of the template strand copied can be achieved analogously to known amplification techniques such as PCR and LCR.
The object of the present invention was therefore to find other DNA polymerases which elongate PNA/DNA primers without having the abovementioned disadvantages.
Surprisingly, it has now been found that temperature-stable DNA polymerases can elongate PNA/DNA primers which, at one end, carry at least one, optionally modified, nucleoside unit with a 3'-hydroxyl group.